Primers for detecting plasmodium

ABSTRACT

The present invention provides an easy and rapid method for detecting/identifying the presence or absence of specific  Plasmodium  parasites and four species of malaria parasites in a human specimen, an anti-malaria measure support system, and a malaria infection-prevention/treatment system, which can contribute to practical diagnosis in a malaria endemic area. According to the present invention, using a genus-specific primer set that can detect four  Plasmodium  parasites that infect humans at a time, and the primer sets each specific to each of four species of  Plasmodium  parasites ( P. falciparum, P. vivax, P. malariae , and  P. ovale ), the presence or absence of infection with these parasites can be detected/identified easily and rapidly.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Divisional of application Ser. No. 12/602,113 filed Feb. 26,2010 (now U.S. Pat. No. 8,309,702), which is a National Stage ofInternational Application No. PCT/JP2008/060115 filed May 27, 2008,claiming priority based on Japanese Patent Application No. 2007-140525,filed May 28, 2007, the contents of all of which are incorporated hereinby reference in their entirety.

TECHNICAL FIELD

The present invention relates to a primer set capable of the rapid andaccurate detection/identification of malaria parasites of the genusPlasmodium in malaria-endemic areas, a method for detecting andidentifying thereof, a detection kit thereof, an anti-malaria measuresupport system, and a malaria infection-prevention/treatment measuresystem.

BACKGROUND ART

In many countries where malaria parasites are endemic, the rapid andaccurate diagnosis of malaria parasites presents challenges. Of fourPlasmodium species, P. falciparum, which can be fatal, must beidentified promptly and distinguished from the other Plasmodium speciesthat produce the disease in humans (Moody, A., Clin. Microbiol. Rev. 15(2002): 66-78).

In addition, most malaria-endemic areas feature infections involving twoor more of these species; these mixed infections often go unrecognizedor underestimated (Zimmerman, P. A., et al., Trends Parasitol. 20(2004): 440-447). Failure to detect mixed infection could result ininadequate treatment, and may result in severe disease (Mayxay, M., etal., Trends Parasitol. 20 (2004): 233-240). There is, therefore, anurgent need to develop malaria diagnostic methods that are operable inendemic areas, easy, rapid, highly sensitive and species-specific.

Currently the easy diagnostic method for malaria is microscopicexamination of blood smears. Given a high density of parasites, suchmicroscopy has relatively high sensitivity and specificity and providesdevelopmental stage and species determination. However, in endemic areaswhere parasite density is generally low, this method is labor-intensive,requires well-trained experts, and may result in therapy being delayed.

To improve the speed and precision of malaria diagnosis in regions wherestandard laboratory diagnosis is not available, researchers havedeveloped rapid diagnostic tests (RDTs) for malaria based onimmunoreaction (Moody, A. Clin. Microbiol. Rev. 15 (2002): 66-78; Ndao,M., et al., J. Clin. Microbiol. 42 (2004): 2694-2700). However, thesensitivity varies between products (Murray, C. K., et al., Trop. Med.Int. Health. 8 (2003): 876-883), and a species-specific product isavailable only for P. falciparum. Very long observation times andconsiderable expertise are required for correct diagnosis by microscopyunder several circumstances: when parasitemia is low, during mixedinfection, after drug treatment, and during the chronic phase of theinfection. Therefore, this situation can lead to false negative resultsor unreliable species diagnosis (Coleman, R., et al., Thailand. Malar.J. 14 (2006): 121).

Subsequently, molecular-biological methods based on DNA amplification,such as nested PCR and real-time quantitative PCR, were developed formalaria diagnosis. Compared to microscopy, these methods havedemonstrated higher sensitivity and greater specificity for mixedinfections (Kimura, K., et al., Parasitol. Int. 46 (1997): 91-95;Perandin, F., et al., J. Clin. Microbiol. 42 (2004): 1214-1219;Rougemont, M., et al., J. Clin. Microbiol. 42 (2004): 5636-5643; Singh,B., et al., Am. J. Trop. Med. Hyg. 60 (1999): 687-692; Singh, B., etal., Lancet. 363 (2004): 1017-1024; Snounou, G., et al., Mol. Biochem.Parasitol. 58 (1993): 283-292; Snounou, G., et al., Mol. Biochem.Parasitol. 61 (1993): 315-320). However, the long turnaround time, highcost, and availability only in well-equipped laboratories render thisPCR technology inadequate for routine diagnosis in the hospitallaboratories and on-site clinics of endemic areas (Hanscheid, T., andGrobusch, M. P., Trends Parasitol. 18 (2002): 395-398).

Regarding malaria detection, Examples 8 and 10 in patent document 1disclose a method of extracting nucleic acids from blood samples andconducting nested PCR to detect four species of Plasmodium. Example 8discloses each forward primer and the reverse primer sequences, whichare different from the primer sequences of the present invention (patentdocument 1).

Patent documents 2 and 4 Patent Publication disclose methods fordetecting one or multiple species of malaria infection based on a solidphase method or nested PCR, in which one or a plurality of multipletypes of primers are used to clinically detect P. falciparum, P. vivax,P. malariae or P. ovale. However, those primers have different primersequences to those of the present invention.

Patent documents 3 Patent Publication discloses a method for detectingP. falciparum and/or P. vivax, in which P. falciparum and/or P. vivaxspecific primers are bound to labelor solid supports. However, thesespecific primer sequences are different from the oligonucleotidesequences of the primer sets of the present invention.

Recently, a novel, easy and highly sensitive technique calledloop-mediated isothermal amplification (LAMP) was developed (Notomi, T.,et al., Nucleic Acids Res. 28 (2000): e63; WO 2000/28082).

LAMP is a nucleic acid amplification method that relies on auto-cyclestrand-displacement DNA synthesis performed by Bst DNA polymerase. Theamplified products are stem-loop structures with several repeatedsequences of the target, and have multiple loops.

The principal merit of this method is that denaturation of the DNAtemplate is not required, (Nagamine, K., et al., Clin. Chem. 47 (2001):1742-1743), and thus the LAMP reaction can be conducted under isothermalconditions (ranging from 60 to 65° C.) LAMP requires only one enzyme andfour types of primers that recognize six distinct target regions. Themethod produces a large amount of amplified product, resulting in easierdetection, such as detection by visual judgment of the turbidity orfluorescence of the reaction mixture (Mori, Y., et al., Biochem.Biophys. Res. Commun. 289 (2001): 150-154). LAMP in which a fluorescentsubstance such as fluorescein, fluorescein isothiocyanate (FITC),X-rhodamine (ROX) or the like is used to measure the fluorescencepolarization values of the reaction mixture, and LAMP in which SYBRGreen 2, a green dye, is used as an intercalator are known (JapaneseUnexamined Patent Publication No. 2002-272475, and WO 2002/103053).

Several investigators have reported LAMP methods for the rapididentification of Plasmodium, Trypanosoma, Babesia, Fusarium, Listeriaand Legionella, and have recommended the usefulness of LAMP assay(Ikadai, H., et al., J. Clin. Microbiol. 42 (2004): 2465-2469; Kuboki,N., et al., J. Clin. Microbiol. 41 (2003): 5517-5524; Thekisoe, O., etal., Mol. Biochem. Parasitol. 122 (2002): 223-236; Japanese UnexaminedPatent Publication No. 2005-245257, Japanese Unexamined PatentPublication No. 2007-61061, Japanese Unexamined Patent Publication No.2003-219878 and Poon, L., et al., Clin. Chem. 52 (2006): 303-306).

Poon et al., estimated that the cost of running a LAMP assay is aboutone tenth that of normal PCR for P. falciparum detection (Poon, L., etal., Clin. Chem. 52 (2006): 303-306). The biggest reduction in cost andtime came from simple sample preparation without previous DNA extraction(Iwasaki, M., et al., Genome Lett. 2 (2003): 119-126).

For the preparation of samples, simply heating the infected blood at 99°C. for 10 minutes was enough to prepare a DNA template for LAMP (Poon,L., et al., Clin. Chem. 52 (2006): 303-306). However, to date, LAMP forthe detection of malaria parasites in clinical diagnosis has beenvalidated only in acute P. falciparum patients (Poon, L., et al., Clin.Chem. 52 (2006): 303-306). Although P. falciparum is the most importantcause of severe disease, its geographic distribution overlaps with thoseof P. vivax, P. malariae and P. ovale infection, and therefore a methodallowing the rapid detection and identification of all four speciesinfecting humans would be desirable.

In recent years, in malaria-endemic areas, the development ofdrug-resistant strains has been a major problem for appropriate malariatreatment. Practicing medical personnel or hospital doctors desire rapidand highly sensitive differentiation methods to obtain information onwhether a patient with a fever is infected with a particular malariaparasite or multiple species of malaria parasites, to therebyappropriately treat said malaria patient with a fever.

-   [Patent document 1] WO2006/88895-   [Patent document 2] Japanese Unexamined Patent Publication No.    1994-261758-   [Patent document 3] Japanese Unexamined Patent Publication No.    1993-227998-   [Patent document 4] Japanese Unexamined Patent Publication No.    2003-250564-   [Non-patent document 1] Poon, L., et al., Clin. Chem. 52 (2006):    303-306

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

To solve the above-mentioned problems, the development of easy and rapidmethods are desired for the detection and identification of the fourspecies of malaria parasites of the genus Plasmodium (particularly, thepresence of mixed infections), which are different from known methodssuch as microscopy or PCR-reaction-mediated methods.

An object of the present invention is to provide a rapid and highlysensitive detection and identification method for clinically detectingand specifying P. falciparum, P. vivax, P. malariae or P. ovale usingLAMP. Further, according to the malaria parasite detection andidentification method of the present invention, using blood samplesobtained at clinics in malaria-endemic areas, a primer set for detectingthe four species of malaria parasites of the genus Plasmodium, where theprimer set has been evaluated through comparison of microscopy and LAMP;a detection method for the four species of malaria parasites of thegenus Plasmodium using the primer set; an identification method; and adetection kit are provided.

Therefore, an object of the present invention is to solve theabove-mentioned problems, and to provide an easy and rapiddetection/identification method for the presence or absence ofPlasmodium parasites or any one of the 4 specific species of malariaparasites in human specimens, which is capable of contributing tomedical care in malaria-endemic areas, and further to provide ananti-malaria measure support system and a malariainfection-prevention/treatment measure system.

Means for Solving the Problems

The present inventors, in aiming to solve such problems, conceived ofusing a Loop-mediated isothermal amplification (LAMP) method, which isan isothermal gene amplification reaction: (WO 00/28082). However, ingeneral, nucleic acid sequences of malaria parasite genes differ greatlyfrom those of other organisms, being rich in AT content. Therefore,existing primer design software was unable to find optimal primer sets,demanding a repetitive process of trial and error with difficulties indesigning each type of primer. Finally, among the many combinations ofsynthetic primer sets, particularly useful primer sets having both highsensitivity and high specificity were found. Thus, it was found that theuse of genus-specific primer sets capable of detecting the fourhuman-infecting species of Plasmodium parasites at the same time andprimer sets each specific to the four species of parasites (P.falciparum, P. vivax, P. malariae and P. ovale) allows the easy andrapid detection/identification of the presence or absence of suchinfections.

It was also found that these results can be effectively used foranti-malaria measure support system and the malariainfection-prevention/treatment measure system can be effectively used.

That is, the present invention can provide the following as described inItems 1 to 27 below.

Item 1. A method for detecting or identifying infection with the genusPlasmodium and/or one or more of Plasmodium falciparum, Plasmodiumvivax, Plasmodium malariae, and Plasmodium ovale in a specimen; themethod comprising the following steps (a) to (c):

a) extracting DNA from the specimen;b) amplifying a particular region of a Plasmodium 18S rRNA gene sequenceby reacting the DNA extracted in the step (a) in a reaction mixturecontaining a strand displacement DNA polymerase and a sequence-specificprimer set; andc) detecting or identifying the presence or absence of an amplifiedproduct of the genus Plasmodium and/or one or more of Plasmodiumfalciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale,amplified in the step (b);

the sequence-specific primer set being an oligonucleotide set containingnucleic acid sequences represented by SEQ ID NOs: 1 to 6 for amplifyinga particular region of a Plasmodium 18S rRNA gene sequence; and/or

one or more of a primer set comprising an oligonucleotide set containingnucleic acid sequences represented by SEQ ID NOs: 7 to 12 for amplifyinga particular region of a Plasmodium falciparum 18S rRNA gene sequence, aprimer set comprising an oligonucleotide set containing nucleic acidsequences represented by SEQ ID NOs: 13 to 18, SEQ ID NOs: 31 to 36, orSEQ ID NOs: 37 to 42 for amplifying a particular region of a Plasmodiumvivax 18S rRNA gene sequence, a primer set comprising an oligonucleotideset containing nucleic acid sequences represented by SEQ ID NOs: 19 to24 for amplifying a particular region of a Plasmodium malariae 18S rRNAgene sequence, and a primer set comprising an oligonucleotide setcontaining nucleic acid sequences represented by SEQ ID NOs: 25 to 30for amplifying a particular region of a Plasmodium ovale 18S rRNA genesequence.

Item 2. A detection or identification method according to Item 1,wherein the DNA extraction from the specimen is carried out by boilingthe specimen containing the DNA, and performing centrifugation.

Item 3. A detection or identification method according to Item 2,wherein the boiling time is from several minutes to ten and severalminutes.

Item 4. A detection or identification method according to any one ofItems 1 to 3, wherein, in the step (b) of amplifying a particular regionof a Plasmodium 18S rRNA gene sequence, the DNA amplification reactionis performed at about 60° C. for about 1 hour using aconstant-temperature water bath or an amplifier specially designed forLAMP.

Item 5. A detection or identification method according to any one ofItems 1 to 4, wherein, in the step (C), the presence or absence of anamplification product of the genus Plasmodium, and/or one or more ofPlasmodium falciparum, Plasmodium vivax, Plasmodium malariae, andPlasmodium ovale is detected or identified using visual observation or areal-time turbidimeter.

Item 6. A detection or identification method according to any one ofItems 1 to 5, which is performed in a malaria endemic area.

Item 7. A detection or identification method according to any one ofItems 1 to 6, wherein infections with the genus Plasmodium and/or one ormore of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae,and Plasmodium ovale are detected or identified simultaneously orseparately.

Item 8. A detection or identification method according to any one ofItems 1 to 6, wherein infection with the genus Plasmodium is detected oridentified using, as the sequence-specific primer set, a primer setcomprising an oligonucleotide set containing nucleic acid sequencesrepresented by SEQ ID NOs: 1 to 6 for amplifying a particular region ofa Plasmodium 18S rRNA gene sequence.

Item 9. A detection or identification method according to any one ofItems 1 to 6, wherein infection with Plasmodium vivax is detected oridentified using, as the sequence-specific primer set, a primer set fordetecting Plasmodium vivax that comprises an oligonucleotide setcontaining nucleic acid sequences represented by SEQ ID NOs: 13 to 18,SEQ ID Nos: 31 to 36, or SEQ ID Nos: 37 to 42 and that is capable ofamplifying a particular region of a Plasmodium vivax 18S rRNA genesequence.

Item 10. A detection or identification method according to any one ofItems 1 to 6, wherein infection with Plasmodium malariae is detected oridentified using, as the sequence-specific primer set, a primer set fordetecting Plasmodium malariae that comprises an oligonucleotide setcontaining nucleic acid sequences represented by SEQ ID NOs: 19 to 24and that is capable of amplifying a particular region of a Plasmodiummalariae 18S rRNA gene sequence.

Item 11. A detection or identification method according to any one ofItems 1 to 6, wherein infection with Plasmodium ovale is detected oridentified using, as the sequence-specific primer set, a primer set forPlasmodium ovale that comprises an oligonucleotide set containingnucleic acid sequences represented by SEQ ID NOs: 25 to 30 and that iscapable of amplifying a particular region of a Plasmodium ovale 18S rRNAgene sequence.

Item 12. A primer set for detecting the genus Plasmodium, comprising anoligonucleotide set containing nucleic acid sequences represented by SEQID NOs: 1 to 6, the primer set being capable of amplifying a particularregion of a Plasmodium 18S rRNA gene sequence.

Item 13. A primer set for detecting Plasmodium vivax, comprising anoligonucleotide set containing nucleic acid sequences represented by SEQID NOs: 13 to 18, SEQ ID NOs: 31 to 36, or SEQ ID NOs: 37 to 42, theprimer set being capable of amplifying a particular region of aPlasmodium vivax 18S rRNA gene sequence.

Item 14. A primer set for detecting Plasmodium malariae, comprising anoligonucleotide set containing nucleic acid sequences represented by SEQID NOs: 19 to 24, the primer set being capable of amplifying aparticular region of a Plasmodium malariae 18S rRNA gene sequence.

Item 15. A primer set for detecting Plasmodium ovale, comprising anoligonucleotide set containing nucleic acid sequences represented by SEQID NOs: 25 to 30, the primer set being capable of detecting a particularregion of a Plasmodium ovale 18S rRNA gene sequence.

Item 16. A primer set for detecting the genus Plasmodium and/or any oneof P. falciparum, P. vivax, P. malariae, and P. ovale, comprising anoligonucleotide primer set containing nucleic acid sequences selectedfrom those defined in Items 12 to 15 and nucleic acid sequencesrepresented by SEQ ID NOs: 7 to 12, the primer set being capable ofamplifying particular regions of 18S rRNA gene sequences of the genusPlasmodium and various species of Plasmodium including a particularregion of a Plasmodium falciparum 18S rRNA gene sequence.

Item 17. A detection kit for the genus Plasmodium and/or any one of P.falciparum, P. vivax, P. malariae, and P. ovale; comprising at least aprimer set selected from the primer sets defined in Items 12 to 16, astrand displacement DNA polymerase, dNTPs and a reaction buffer.

Item 18. A detection kit according to Item 17, wherein the detection kitdetects the genus Plasmodium and/or P. falciparum, P. vivax, P.malariae, and P. ovale, simultaneously or separately.

Item 19. An anti-malaria measure support system comprising:

means for inputting and storing malaria-infected patient informationincluding the number positive for the genus Plasmodium parasites thatcause malaria in a malaria endemic area, and the carrier rate in thearea;

a malaria infection-prevention public health measure guide database thatspecifies public health measures for the malaria endemic area based onthe malaria-infected patient information, into which database publichealth measure selection information for a malaria parasite-infectedarea to be inputted together with the measure priority indicating whichof the public health measures should be given priority in selection hasbeen inputted;

a public health measure extraction section that extracts public healthmeasures for the malaria-infected endemic area and the priority thereoffrom the malaria infection-prevention public health measure guidedatabase, according to malaria-infected patient information aboutmalaria parasites in a subject; and

a public health measure display section that displays the public healthmeasures extracted in the public health measure extraction section,together with the priority thereof.

Item 20. An anti-malaria measure support system according to Item 19,wherein the malaria-infected patient information about malaria parasitesin the malaria endemic area is obtained by identifying the presence orabsence of infection with the genus Plasmodium using a detection oridentification method according to Item 1 and/or a primer set accordingto Item 12, or the Plasmodium detection kit according to Item 17.

Item 21. An anti-malaria measure support system comprising:

means for inputting and storing malaria parasite therapeutic agentinformation for specifying a malaria therapeutic agent that acts oninfection with one or a plurality of four species of malaria parasites;

patient information input means for inputting and storing patientinformation including information about the pathogen of malariainfection in a patient with a fever in a malaria endemic area;

patient information input means for inputting and storing patientinformation including information about the pathogen of malariainfection in a patient with a fever in a malaria non-endemic area;

a treatment guide database into which, according to indices of efficacyagainst malaria parasites detected in a subject, malaria parasitetherapeutic agent selection information to be inputted together with apriority that indicates which malaria therapeutic agent should be givenpriority in selection for use against the malaria parasites;

a malaria therapeutic agent extraction section that extracts, accordingto the information about the pathogen of malaria infection in thesubject, malaria therapeutic agents to be administered and the prioritythereof, from the treatment guide database; and

a malaria therapeutic agent display section that displays the malariatherapeutic agents extracted in the above malaria therapeutic agentextraction section, together with the priority thereof.

Item 22. An anti-malaria measure support system according to Item 21,wherein the information about the pathogen of malaria infection in apatient with a fever is obtained by identifying infection with one or aplurality of four species of malaria parasites using a detection oridentification method according to any one of Items 1 to 11 and/or aprimer set according to any one of Items 12 to 16, or a detection kitaccording to Item 17 or 18.

Item 23. An anti-malaria measure support system in which a public healthmeasure relating to an anti-malaria measure and a malaria parasitetreatment measure relating to an anti-malaria measure are carried out incombination;

the public health measure comprising:

means for inputting and storing malaria-infected patient informationincluding the number positive for the genus Plasmodium that causesmalaria in a malaria endemic area, and the carrier rate in the area;

a malaria infection-prevention public health measure guide database thatspecifies public health measures for the malaria endemic area based onthe malaria-infected patient information, into which database publichealth measure selection information for a malaria parasite-infectedarea to be inputted together with a priority of measures that indicateswhich of the public health measures should be given priority inselection has been inputted;

a public health measure extraction section that extracts public healthmeasures for the malaria-infected endemic area and the priority thereof,from the malaria infection-prevention public health measure guidedatabase according to malaria-infected patient information about malariaparasites in the subject; and

a public health measure display section that displays the public healthmeasures extracted in the public health measure extraction section,together with the priority thereof; and

the malaria parasite treatment measure comprising:

means for inputting and storing malaria parasite therapeutic agentinformation for specifying a malaria therapeutic agent that acts oninfection with one or a plurality of four species of malaria parasites;

patient information input means for inputting and storing patientinformation including information about the pathogen of malariainfection in a patient with a fever in the malaria endemic area;

patient information input means for inputting and storing patientinformation including information about the pathogen of malariainfection in a patient with a fever in a malaria non-endemic area;

a treatment guide database into which, according to indices of efficacyagainst malaria parasites detected in a specimen, malaria parasitetherapeutic agent selection information to be inputted together with apriority that indicates which malaria therapeutic agent should be givenpriority in selection for use against the malaria parasites;

a malaria therapeutic agent extraction section that extracts, accordingto the information about the pathogen of malaria infection in thesubject, malaria therapeutic agents to be administered and the prioritythereof, from the treatment guide database; and

a malaria therapeutic agent display section that displays the malariatherapeutic agents extracted in the above malaria therapeutic agentextraction section, together with the priority thereof.

Item 24. An anti-malaria measure support system according to Item 23,wherein the malaria-infected patient information about malaria parasitesin the malaria endemic area is obtained by identifying the presence orabsence of infection with the genus Plasmodium using a detection oridentification method according to Item 1 and/or a primer set accordingto Item 12, or the genus Plasmodium detection kit according to Item 17or 18; and/or information about the pathogen of malaria infection in apatient with a fever is obtained by identifying infection with one or aplurality of four species of malaria parasites using a detection oridentification method according to any one of Items 1 to 11 and/or aprimer set according to Item 16, or a Plasmodium species detection kitaccording to Item 17 or 18.

Item 25. A malaria infection-prevention measure system for persons whoplan to travel to a malaria endemic area, the system comprising:

means for obtaining the state of the implementation of public healthmeasures in the malaria endemic area, information about the pathogens ofmalaria infection in the endemic area, and the state of the treatment ofinfected patients, from the anti-malaria measure support systemaccording to Item 23;

means for selecting a malaria prophylactic/therapeutic agent from amalaria parasite treatment guide database; and

means for administering the selected agent before travel.

Item 26. A malaria infection-prevention/treatment measure system forreturnees from a malaria endemic area, the system comprising:

means for obtaining the state of the implementation of public healthmeasures in the malaria endemic area, information about the pathogens ofmalaria infection in the endemic area, and the state of the treatment ofinfected patients;

means for selecting a malaria prophylactic/therapeutic agent from amalaria parasite treatment guide database; and

means for administering the selected agent after returning from themalaria endemic area, according to Item 23.

Item 27. A malaria infection-prevention/treatment measure systemaccording to Item 26, wherein, when a returnee from the malaria endemicarea has a fever, identification of Plasmodium falciparum, Plasmodiumvivax, Plasmodium malariae, or Plasmodium ovale is performed to selectand administer a malaria therapeutic agent that should be given priorityin selection.

Use of one of the primers or primer set of the above Items (1) to (5)for LAMP allows the annealing of a particular region of the genusPlasmodium 18S rRNA gene, a P. falciparum 18S rRNA gene, a P. vivax 18SrRNA gene, a P. malariae 18S rRNA gene or a P. ovale 18S rRNA gene.Amplifying this under the amplification conditions of the LAMP methodallows amplification of a specific gene region. The presence or absenceof such an amplification product is analyzed by electrophoresis or aneasy detection method. In such a method, infections by the specificgenus Plasmodium and each of the four species of malaria parasites canbe simultaneously or separately detected and differentiated.

When the detection method of the present invention is used for specificspecimens (for example, human blood), DNA samples can be isolated fromthe specimens, with these DNA samples, any one of the primer sets ofItem (1) to Item (5) were reacted for amplification, thereby confirmingthe presence or absence of any amplified DNA products. In such a way,any of the genus Plasmodium or the four species of malaria parasites: P.falciparum, P. vivax, P. malariae or P. ovale can be detectedsimultaneously or separately.

Effects of the Invention

The present invention allows the use of a primer set for LAMP, whereinthe primer set comprises an oligonucleotide set containing the nucleicacid sequences of SEQ ID NOs: 1 to 6, 7 to 12, 13 to 18 (31 to 36 or 37to 42), 19 to 24 or 25 to 30; and allows the simultaneous or separateamplification of a common region of the genus Plasmodium 18S rRNA genes,or a particular region of the 18S rRNA genes of each of the four speciesof human malaria parasites: P. falciparum, P. vivax, P. malariae or P.ovale; thereby allowing simultaneous or separate detection ordifferentiation of the presence or absence of any human malaria parasiteinfection or one of the four species of human malaria parasites.

The method for detecting or differentiating the presence or absence ofmalaria parasite infection or one of the four species of human malariaparasites of the present invention comprises: amplifying DNA samplesobtained from specimens by LAMP (isothermal gene amplification) using aprimer set comprising the oligonucleotide set containing the nucleicacid sequences of SEQ ID NOs: 1 to 6, 7 to 12, 13 to 18 (31 to 36 or 37to 42), 19 to 24 or 25 to 30; and analyzing the presence or absence ofany amplification products. Such a detection or differentiation methodallows easy, rapid and reliable detection or differentiation of thepresence or absence of any malaria parasite infections, or one of thefour species of malaria parasites: P. falciparum, P. vivax, P. malariaeor P. ovale simultaneously or separately. The present invention alsoprovides a kit for the simultaneous or separate detection oridentification of such human malaria parasites or the four species ofmalaria parasites.

The development of drug-resistant strains has become a major issue forappropriately treating malaria. The method for simultaneouslydifferentiating the four species of malaria parasites: P. falciparum, P.vivax, P. malariae and P. ovale of the present invention provides, inmalaria-endemic regions, practicing medical personnel or hospitaldoctors with rapid and highly sensitive information on whether a patientwith a fever is infected with a particular malaria parasite or multiplemalaria parasites, allowing rapid and appropriate treatment for amalaria patient with a fever.

The use of the genus Plasmodium or the four species of malaria parasitedetection/identification method of the present invention makes itpossible to monitor the therapeutic effects of malaria therapeutic agentadministration to malaria-infected patients.

According to the anti-malaria measure support system of the presentinvention, clinical practitioners and hospital doctors can checkinformation about the pathogen of malaria infection in a patient with afever against the malaria therapeutic agent guide database in a PC orvia a cellular phone to have a conventional software to operate, therebyobtaining a display of which malaria therapeutic agent should be givenpriority in selection, or which malaria therapeutic agents should beused in combination.

Using the malaria infection-prevention measure system of the presentinvention, it is possible for a person who plan to travel to a malariaendemic area to know beforehand the malaria infections that are endemicin the area and the state of appearance of drug-resistant strains. Thus,to prevent malaria infection, the person can take, before travel, apreferable malaria therapeutic agent to be given priority in selectionfor use against the malaria infections that are endemic in the area, sothat even if the person should be infected with malaria, symptoms due tomalaria infection, such as a fever, can be reduced at an early stage andserious conditions can be prevented, making it possible to exterminatemalaria parasites from the person's blood at an early stage.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the locations of LAMP targets and priming sites forPlasmodium genus (A) and four Plasmodium species (B), and the 18S rRNAgene nucleotide sequences. (A) The locations of the priming sites by thePlasmodium genus-specific primer set in the reference sequence (GenBankAccession No. M19173.1) are indicated by arrows. (B) Partial sequencealignment of the 18S rRNA genes of four human malaria parasites, P.falciparum (Pf; GenBank Accession No. M19173.1), P. vivax (Pv; GenBankAccession no. U03079), P. malariae (Pm; GenBank Accession No. M54897),and P. ovale (Po; GenBank Accession No. L48986), along with thespecies-specific primer annealing sites.

FIG. 2 is a schematic diagram of the anti-malaria measure support systemusing LAMP.

FIG. 3 is a flow chart of the process of the anti-malaria measuresupport system based on the principle of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Preferred embodiments of the present invention are explained in detailbelow.

The present invention is an easy and highly reliable detection systemfor routine malaria parasite screening, both at hospitals andlaboratories, or at malaria clinics in endemic areas. For example, thegenus Plasmodium or any one of the four species of malaria parasites: P.falciparum, P. vivax, P. malariae or P. ovale when present in humanblood samples are determined by an isothermal gene amplification methodthat employs LAMP using oligonucleotide primers specific to the genusPlasmodium and each one of the four species of malaria parasites.Specifically, the invention is based on a method comprising the steps oftargeting a particular region of the genus specific Plasmodium 18S rRNAgene sequence, the P. falciparum 18S rRNA gene sequence, P. vivax 18SrRNA gene sequence, P. malariae 18S rRNA gene sequence, or P. ovale 18SrRNA gene sequence; amplifying the particular region of the Plasmodium18S rRNA gene sequence, the P. falciparum 18S rRNA gene sequence, P.vivax 18S rRNA gene sequence, P. malariae 18S rRNA gene sequence or P.ovale 18S rRNA gene sequence using the primer set of Item 16; andanalyzing the presence or absence of any amplification products.

The detection/identification method of the present invention can beapplied to malaria parasites present in human blood that is infectedwith malaria parasites.

As used herein, the term “specimen” may include the genus Plasmodium orone of the four specific species of malaria parasites: P. falciparum, P.vivax, P. malariae or P. ovale, implying human blood samples to betargeted by the detection/identification method of the presentinvention. As used herein, the term “detection” implies, for example,determining whether malaria parasites present in a blood sample aremalaria parasites of a specific Plasmodium species, and is sometimesused as a synonym for determination.

As used herein, the term “identification” sometimes impliesdistinguishing between specific Plasmodium species such as P.falciparum, P. vivax, P. malariae or P. ovale and the other Plasmodiumspecies; and detecting simultaneously or separately; in specimens whereat least one species of Plasmodium species is present. However, the term“identification” generally implies identifying a particular malariaparasite to be detected amongst multiple malaria parasites. Hence, theterm “identification” includes the detection of single malaria parasiteinfections and multiple malaria parasite infections.

The LAMP method used in the present invention is a gene amplificationmethod in which, unlike PCR, thermoregulation (thermal cycle) is notrequired in the amplification steps, one kind of DNA polymerase is used,and genes are amplified at a constant temperature (isothermaltemperature) (WO 2000/28082 and Notomi, T., et al., Nucleic Acids Res.28 (2000): e63).

The above DNA polymerase may be any template-dependent nucleic acidsynthetic enzyme that processes strand displacement activity. Suchenzymes include Bst DNA polymerase (Large Fragment), Bca (exo-) DNApolymerase, the Klenow fragment of Escherichia coli DNA polymerase I,Vent (Exo-) DNA polymerase (Vent DNA polymerase without exonucleaseactivity), DeepVent (Exo-) DNA polymerase (DeepVent DNA polymerasewithout exonuclease), KOD DNA polymerase and the like.

Bst DNA polymerase (Large Fragment) is preferably used. When this enzymeis used, the reaction is preferably performed around at 65° C., which isan optimum temperature for the enzyme reaction.

An amplified product of the LAMP method can be detected by knowntechniques. For example, it can be detected using a labeledoligonucleotide that specifically recognizes the amplified genesequence; or the reaction mixture can be directly subjected to agaroseelectrophoresis after completion of the reaction for easy detection. TheLAMP method produces a ladder of multiple bands having different baselengths.

Furthermore, the white suspension caused by magnesium pyrophosphateaccumulation as a by-product of amplification can be detected by thenaked eye or with a turbidimeter (Mori, Y., et al. Biochem. Biophys.Res. Commun. 289 (2001): 150-154).

Alternatively, amplification can be simply inspected with the naked eyeusing SYBR Green I (product of Applied Biosystems), which turns green inthe presence of amplified DNA. However, SYBR Green I results wereconsistent with those deduced from a real-time turbidimeter (Parida, M.,et al., J. Clin. Microbiol. 43 (2005): 2895-2903 and Japanese UnexaminedPatent Publication No. 2001-242169). Since the turbidity assay can becarried out in a closed system, the risk of contamination is lower thanthat for agarose gel electrophoresis. This is an additional merit ofLAMP in clinical use (Enosawa, M., et al. J. Clin. Microbiol. 41 (2003):4359-4365; Seki, M., et al., J. Clin. Microbiol. 43 (2005): 1581-1586;Poon, L., et al., Clin. Chem. 52 (2006): 303-306).

Thus, LAMP diagnosis, in principle, does not require expensive reagentsfor DNA extraction, a turbidimeter, a thermal cycler, or a skilledtechnician). The template can be prepared by direct heat-treatment ofblood samples, without time-consuming and expensive DNA extractionsusing a commercial kit.

For example, in the field in malaria-endemic areas, only boiling andseparating a very small amount of blood from a subject's fingertipallows the preparation of a desired DNA sample enough for the LAMPreaction.

Moreover, LAMP requires only a simple incubator, such as a heat block ora water-bath that provides a constant 60° C., which makes it moreeconomical and practical than nested PCR or real-time PCR.

A genus specific primer set common to the four species of human malariaparasite 18S rRNA genes, and each specific primer set of the presentinvention for the P. vivax 18S rRNA gene, P. malariae 18S rRNA gene, andP. ovale 18S rRNA gene, target particular regions of each 18S rRNA gene.The primer sets are designed to have a total of 4 kinds of primer as aset, in which two are loop forming 2 kinds of inner primers:(FIP(F1c-F2) and BIP(B1-B2c)), and the other two are 2 kinds of outerprimers (F3, B3c). Particular regions of each amplified gene rangebetween about 70 to 500 base pair length.

The inner primers amplify the nucleic acid sequence of the targetregion, and are characterized by including: (a) as a first segment, anucleic acid sequence that anneals the target gene and functions as aprimer; and (b) as a second segment, a nucleic acid sequence that iscomplementarily to the 3′ nucleic acid sequence of the first segment,and positions at the 5′ side of the first segment.

Further, use of loop primers (LPB, LPF) having a complementary sequenceto the single strand portion of the dumbbell structure's 5′ terminusloop, where the dumbbell structure functions as the origin of theamplification reaction, can increase the number of origins in DNAsynthesis. Therefore, use of loop primers can increase amplificationefficacy, and shorten the time required for amplification to about onethird to a half. Outer primers recognize the 3′ terminus nucleic acidsequence of the target region, and have a nucleic acid sequencefunctioning as the origin of the synthesis.

The inventors attempted to design each inner primer ((FIP(FIc-F2),BIP(B1-B2c)), outer primer (F3, B3c) and loop primer (LPB, LPF) usingthe LAMP designing software, PRIMEREXPLORER V3 (product of FujitsuLimited), based on a genus-specific nucleic acid sequence of the 18SrRNA genes common to the four species of human malaria parasites, andeach species-specific nucleic acid sequence of the 18S rRNA genes of P.falciparum, P. vivax, P. malariae and P. ovale. However, in general, thenucleic acid sequences of malaria parasite genes differ greatly fromthose of other organisms, being rich in AT content. Therefore, existingprimer design software was unable to find optimal primer sets, demandinga repetitive trial-and-error process, and causing difficulties indesigning each type of primer. Finally, among many combinations of suchsynthesized primer sets, usable primer sets both high in sensitivity andspecificity were found. Thus, the inventors have successfully designed agenus-specific primer capable of detecting the four species ofhuman-infectious malaria parasites at the same time, and specificprimers for each of the four species of malaria parasites (P.falciparum, P. vivax, P. malariae and P. ovale) from each 18S rRNA gene.

Once the nucleic acid sequence of a primer oligonucleotide isdetermined, the oligonucleotide can be synthesized by known means, forexample, by an automatic DNA synthesizer produced by PerkinElmer, Inc.

According to the present invention, examples of a plasmodiumgenus-specific primer set specific to a nucleic acid sequence of the 18SrRNA genes common among the four species of human malaria parasites, andspecific primer sets for each of the malaria parasite 18S rRNA gene ofP. falciparum, P. vivax, P. malariae and P. ovale include, for example,seven primer sets as follow primer sets for the genus plasmodium and thefour species of malaria parasites:

A primer set for the genus Plasmodium [F3(SEQ ID NO: 1), B3c(SEQ ID NO:2), FIP(F1c-F2)(SEQ ID NO: 3), BIP(B1-B2c)(SEQ ID NO: 4), LPF(SEQ ID NO:5), LPB(SEQ ID NO: 6)];A primer set for P. falciparum [F3(SEQ ID NO: 7), B3c(SEQ ID NO: 8),FIP(F1c-F2)(SEQ ID NO: 9), BIP(B1-B2c)(SEQ ID NO: 10), LPF(SEQ ID NO:11), LPB(SEQ ID NO: 12)];A primer set for P. vivax [F3(SEQ ID NO: 13), B3c(SEQ ID NO: 14),FIP(F1c-F2)(SEQ ID NO: 15), BIP(B1-B2c)(SEQ ID NO: 16), LPF(SEQ ID NO:17), LPB(SEQ ID NO: 18)]; [PvFIP-9 (F1c+F2)(SEQ ID NO: 31),PvBIP-9(B1+B2c) (SEQ ID NO: 32), PvF3-9 (SEQ ID NO: 33), PvB3c-9 (SEQ IDNO: 34), PvLPF-9 (SEQ ID NO: 35), PvLPB-9 (SEQ ID NO: 36)]; or[PvFIP-7(F1c+F2) (SEQ ID NO: 37), PvBIP-7(B1+B2c) (SEQ ID NO: 38),PvF3-7(SEQ ID NO: 39), PvB3c-7 (SEQ ID NO: 40), PvLPF-7 (SEQ ID NO: 41),PvLPB-7(SEQ ID NO: 42)];A primer set for P. malariae [F3(SEQ ID NO: 19), B3c(SEQ ID NO: 20),FIP(F1c-F2)(SEQ ID NO: 21), BIP(B1-B2c)(SEQ ID NO: 22), LPF(SEQ ID NO:23), LPB(SEQ ID NO: 24)]; andA primer set for P. ovale [F3(SEQ ID NO: 25), B3c(SEQ ID NO: 26),FIP(F1c-F2)(SEQ ID NO: 27), BIP(B1-B2c)(SEQ ID NO: 28), LPF(SEQ ID NO:29), LPB(SEQ ID NO: 30)].

Here, primers each specific to the genus Plasmodium, P. falciparum, P.vivax, P. malariae and P. ovale are primers capable of specificallyamplifying a particular region of the 18S rRNA genes common to saidPlasmodium species and each particular region of each 18S rRNA gene ofP. falciparum, P. vivax, P. malariae and P. ovale.

Examples of sequences characterized by the genus Plasmodium and the fourspecies of malaria parasites subjected to detection/identification usingLAMP of the present invention include the 18S rRNA gene sequences of P.falciparum (P. falciparum: GenBank Accession No. M19173.1, M19173.2,M19172), P. vivax (P. vivax: GenBank Accession No. U03079, U03080,X13926), P. malariae (P. malariae: GenBank Accession No. M54897), and P.ovale (P. ovale: GenBank Accession No. L48986, L48987) deposited atGenBank.

The detection or identification of the genus Plasmodium or one of thefour species of malaria parasites present in the specimen are conductedby isothermal gene amplification generally in the range of at 60 to 65°C. for 15 minutes to 1 hour, using at least one set of theaforementioned five primer sets, following the procedure of LAMP. Thatis, DNA collected from specimens such as blood samples and the like isisolated by a known method, and this DNA is amplified using said primerset. The presence of the amplified DNA product can be easily detected byLAMP, or by a general method of electrophoresis.

The aforementioned easy detection includes: 1) visual inspection of theamplification reaction mixture for white turbidity (WO 2001/83817); 2) amethod for measuring the fluorescence polarization values of thereaction mixture using a fluorescent substance such as fluorescein,fluorescein isothiocyanate (FITC), X-rhodamine (ROX) or the like(Japanese Unexamined Patent Publication No. 2002-272475), which uses acontinuous fluorometer such as an ABI Prism 7700 (product of AppliedBiosystems) and the like, allowing for the confirmation of amplificationor kinetic analysis; and 3) visual inspection using SYBR Green 2, whichuses a fluorescent green dye as an intercalator (WO 2002/103053). By anyof these methods, the presence or absence of any amplification products(presence or absence of the target 18S rRNA gene) can be inspected usingthe naked eye.

According to the present invention, a malaria parasite detection kitcapable of detecting any of the genus Plasmodium, P. falciparum, P.vivax, P. malariae or P. ovale simultaneously or separately can beprovided.

The above malaria parasite detection kit can be prepackaged with varioustypes of reagents necessary for detecting nucleic acid amplified usingthe primer set of the present invention. Specifically, various types ofoligonucleotides necessary for the primers or loop primers of thepresent invention, four species of dNTPs as substrates for nucleic acidsynthesis, a template-dependent nucleic acid synthase with stranddisplacement activity, a buffer or a salt to provide preferableconditions for enzymatic reaction, a protective agent for stabilizingenzymes or templates, and, when indicated, reagent(s) necessary fordetecting a reaction product are provided as a kit.

Example of Kit Components:

(1) A reaction mixture containing a primer set for the genus Plasmodium[F3 (SEQ ID NO: 1), B3c (SEQ ID NO: 2), FIP (F1c-F2)(SEQ ID NO: 3),BIP(B1-B2c) (SEQ ID NO: 4), LPF (SEQ ID NO: 5), LPB (SEQ ID NO: 6)];(2) A reagent for the visual detection of fluorescence;(3) An enzyme mixture solution (including Bst DNA polymerase);(4) A positive control (for the genus Plasmodium); and(5) Distilled water are provided.

The present invention further provides an anti-malaria measure supportsystem and a malaria infection-prevention/treatment measure system.

Malaria-infected patient information includes the number positive forthe genus Plasmodium that causes malaria in a malaria endemic area, andthe carrier rate in the area; and specifically, the informationincludes, as basic information, subject information such as the name,sex, age, weight, pregnancy or non-pregnancy status, family structure,residential address, birthplace, names of pre-existing diseases, namesof drugs being taken, history of drug side effects, etc., of individualsubjects residing in the endemic area, together with their positivity ornegativity for the genus Plasmodium and the acquisition ornon-acquisition of resistance to malaria therapeutic agents, and themalaria parasite carrier rate in the area. The means for inputting andstoring malaria-infected patient information are, for example, computerinput devices and storage devices.

The carrier rate for the genus Plasmodium that causes malaria in amalaria endemic area is expressed as a percentage obtained by dividingthe number of subjects positive for the genus Plasmodium by the numberof subjects of the genus Plasmodium detection test and multiplying theresultant quotient by 100. In the anti-malaria measure support system ofthe present invention, for example, a genus Plasmodium detection methodusing LAMP can be particularly preferably used in malaria endemic areas.In a subject who has resided in a malaria endemic area for a long periodand has acquired immunity (resistance) against malaria, the number ofparasites in the subject's blood is as small as about one hundredth toabout one thousandth of that in a patient with malarial fever.Microscopic detection of such a small number of parasites with highsensitivity is very difficult. Also, after malaria infection and thelatent period, in the early stages of the period in which a feverdevelops due to the appearance of malaria parasites in the blood, thenumber of parasites is small and therefore microscopic detection of theparasites is extremely difficult. Microscopic detection with highsensitivity is also very difficult when a malaria therapeutic agent hasalready been administered and the number of parasites in the subject'sblood has been remarkably reduced by the effects of the therapeuticagent.

In many malaria endemic areas, satisfactory DNA extractors do not existor are not provided. In the preparation of DNA for PCR, when highly pureDNA cannot be obtained using an extraction kit, PCR cannot be performedbecause PCR does not occur due to the inhibitor of the DNA amplificationenzymes for PCR, which is present in blood.

In the genus Plasmodium detection method used in the anti-malariameasure support system of the present invention, the DNA sample can bevery easily obtained by, for example, collecting a very small amount ofblood from the subject's finger tip, boiling the blood in boiling waterfor 10 minutes, and centrifuging the blood at 10000 rpm for 1 minute toobtain the supernatant, which can be used as DNA. Therefore, the methodin which the genus Plasmodium in a specimen are detected using the genusPlasmodium detection primer set of the present invention, which canamplify a specific region of the 18S rRNA gene sequence of Plasmodium,can be advantageously used in malaria endemic areas. Further, whenresearch is conducted not only on subjects in a malaria endemic area,but also on the rate of mosquitoes collected in an endemic area thatcarry malaria parasites to obtain important data for malaria epidemicprediction based on research on mosquitoes carrying malaria parasites,since DNA for use in the method of the present invention can be moreeasily extracted from mosquitoes than DNA for PCR, the frequency of thedetection of the genus Plasmodium in specimens, obtained using a genusPlasmodium detection primer set of the present invention, which canamplify a specific region of the 18S rRNA gene sequence of Plasmodium,can be stored in a storage device as a piece of information for malariaepidemic prediction in the malaria infection-prevention public healthmeasure database.

The method for detecting or identifying the genus Plasmodium or fourspecies of malaria parasites according to the present invention issimpler, less expensive, more easily operable, and has highersensitivity and higher specificity, than microscopy and PCR, andtherefore can be used most preferably in endemic areas.

Public health measures for malaria infection endemic areas based onmalaria-infected patient information include the following (1) to (4):(1) eliminating, from the area, environments where mosquitoes are likelyto breed, and eliminating sources of mosquitoes by exterminatingmosquitoes and mosquito larvae bred in stagnant water; (2) spraying aninsecticide in houses, sheds, etc., providing doors and windows withwire screens, and preferably, installing air-conditioners in houses ifthe residents of the houses can afford them; (3) providing beds withmosquito nets (mosquito nets with long-lasting effects: LLIN, SumitomoChemical Co., Ltd.) impregnated with an insecticide (pyrethroid-basedinsecticide: permethrin), applying to human skin an insect repellentspray containing an insect repellent (diethyltoluamide: DEET), andtaking measures such as wearing long-sleeved shirts after sunset,spraying clothes with an insecticide, etc.; and (4) prophylacticallyadministering a therapeutic agent, such as a mixture of mefloquine andartesunate, chloroquine, artemisinin, quinine, doxycycline, or the like,based on information about malaria parasites occurring with highfrequency in the endemic area and drug-resistant strains, so that themalaria therapeutic agent also serves as a malaria prophylactic agent.

For infection-positive patients, malaria parasite extermination(treatment) with a malaria therapeutic agent is taken intoconsideration. Information about malaria parasites occurring with highfrequency in the endemic area and drug-resistant strains is availablefrom the websites of the departments of health of countries with endemicareas; the Japan Health Sciences Foundation, policy innovative drugdevelopment general research project, research group on chemotherapy oftropical diseases, the Guidance for Parasitic Disease Chemotherapy,revised version 6.0 (2007); and the guidelines of the Expert Meeting onMalaria Chemoprophylaxis published on March, 2005. The malariatherapeutic agent can be easily selected according to the species ofmalaria parasites detected or identified by the method of the presentinvention, based on known standards for selecting therapeutic agents.

The priority of measures, which indicates which of the above publichealth measures (1) to (4) should be given priority in selection, isdetermined considering the degree of malaria prevalence in the area,economic conditions of the country, environmental and economicconditions of the residents, etc. The measure (1), which can be taken bythe country, local government, autonomous body, or the like, should begiven priority, and then the measures (2) and (3), which can be easilytaken by individual subjects and other residents, are desirable.

The measure (4), prophylactic administration of a therapeutic agent, hasa lower priority considering the general economic conditions ofresidents in malaria endemic areas, and is likely to be limited toresidents with special needs, such as infants, pregnant women, etc. Themeasure (4) is not sufficient at present. The priority of measuresvaries depending on the endemic area, and may be, for example,preferably (1)>(2)=(3)>(4), and from a practical point of view,(2)=(3)>(4)>(1). Such measure priority is extracted in a public healthmeasure extraction section (e.g., a program) in view of the degree ofmalaria prevalence, economic conditions of the country, environmentaland economic conditions of the residents, etc., and is displayed on apublic health measure display section (e.g., display).

The information stored in the malaria infection-prevention public healthmeasure guide database, into which the public health measure selectioninformation for the inputted malaria parasite-infected area has beeninputted, may include, as described above, the type and frequency ofmalaria that is actually endemic in the area, information aboutdrug-resistant strains, information about the public health measures (1)to (4), instruction information about the measures, malaria epidemicprediction, malaria diagnosis methods, and information about drugsselected for respective species of infecting malaria parasites.

Using DNA extracted from a subject, the malaria parasite detectionmethod of the present invention is carried out to obtain infectedpatient information about the presence or absence of malaria infectionin subjects;

and this information is checked against the public health measures forthe malaria infection endemic area, and their priority, obtained fromthe public health measure extraction section that extracts the priorityof the public health measures from the malaria infection-preventionpublic health measure guide database, and the public health measuredisplay section that displays the public health measures extracted inthe public health measure extraction section together with theirpriority. Thus, the public health measures that should be taken by localgovernments or individual subjects in the endemic area can be carriedout sequentially or simultaneously. Further, the effects of such publichealth measures can be stored as information in the malariainfection-prevention public health measure guide database. This canupdate the malaria infection-prevention public health measure guidedatabase. The anti-malaria measure support system of the presentinvention can thus be provided to a malaria endemic area.

The subject information including information about the presence orabsence of infection with malaria parasites in subjects can be managedand obtained by a conventional technique using a PC (personal computer)or a cellular phone, a facsimile, etc. The information to be stored inthe malaria infection-prevention public health measure guide databasecan be obtained from Internet websites using a PC or a cellular phone,or from brochures.

In the anti-malaria measure support system of the present invention, themeans for inputting and storing malaria therapeutic agent informationfor specifying a malaria therapeutic agent that acts on infection withone or a plurality of four species of malaria parasites indicates meansfor storing information including the effects of drugs, drug names,generic names, areas in which respective drugs are preferably applied,administration periods, dosages, types and frequencies of side effects,severity of side effects, contraindications, information about infantsand pregnant women, information about concomitant use with other drugs,drug prices, etc., into the “treatment guide database”, considering theactual appearance of drug-resistant strains in each malaria endemicarea, and the degree thereof, or considering the clinical symptoms ofeach type of malaria, age, and pregnancy or non-pregnancy status of thepatient.

It is important that patients with a fever caused by malaria infectionbe differentially diagnosed from patients with other infections such asinfluenza, since malaria infection, and in particular P. falciparuminfection, results in a serious outcome if treatment is delayed.Therefore, prompt, highly sensitive, and highly specific differentialdiagnosis is desired. In particular, as described above, satisfactoryDNA extractors do not exist or are not provided in many malaria endemicareas, and in the early stages of a fever, the number of malariaparasites is so small that microscopic identification of malariaparasites is not easy.

The method for identifying P. falciparum, P. vivax, P. malariae, or P.ovale using a primer set comprising a sequence specific to P.falciparum, P. vivax, P. malariae, or P. ovale according to the presentinvention, or the identification/detection kit therefor, makes itpossible to identify infection with one or a plurality of four speciesof malaria parasites more easily, more rapidly, and with highersensitivity and higher specificity, than other identification methodssuch as PCR and microscopy.

Further, the malaria parasite detection/identification method of thepresent invention enables monitoring of the therapeutic effects of theadministration of malaria therapeutic agents to malaria-infectedpatients.

The system of the present invention is based on the malariaparasite-specific detection or identification technique. The “patientinformation input means for inputting and storing patient informationincluding information about the pathogen of malaria infection in apatient with a fever in a malaria endemic area” is means for inputtingand memorizing/storing subject information including information aboutthe pathogen of malaria infection in a patient with a fever in a malariaendemic area, and the name, age, sex, weight, pregnancy or non-pregnancystatus, family structure, residential address, birthplace, names ofpre-existing diseases, names of the drugs being taken, drug side-effecthistory, etc. of the patient with a fever, into a PC (personalcomputer). The input and memorizing/storage can be performed from a PCor a cellular phone, or as facsimile information from medical facilitiesand hospitals, based on information obtained from endemic areas.

The “patient information input means for inputting and storing patientinformation including information about the pathogen of malariainfection in a patient with a fever in a malaria non-endemic area” isthe same as the “patient information input means for inputting andstoring patient information including information about the pathogen ofmalaria infection in a patient with a fever in a malaria endemic area”except that the patient with a fever is “a patient with a fever in amalaria non-endemic area”.

In the above, “comprising a treatment guide database into which,according to indices of efficacy against malaria parasites detected in asubject, malaria therapeutic agent selection information to be inputtedtogether with a priority that indicates which malaria therapeutic agentshould be given priority in selection for use against the malariaparasites; a malaria therapeutic agent extraction section that extracts,according to the information about the pathogen of malaria infection inthe subject, malaria therapeutic agents to be administered and thepriority thereof, from the treatment guide database; and a malariatherapeutic agent display section that displays the malaria therapeuticagents extracted in the above malaria therapeutic agent extractionsection, together with the priority thereof” indicates a process inwhich a clinical practitioner or a hospital doctor who has obtainedinformation about the pathogens of malaria infection in a patient with afever in a malaria endemic or non-endemic area displays which malariatherapeutic agent should be given priority in selection for use againstthe infecting malaria parasites, considering the species of malariaparasites infecting the patient with a fever, the symptoms of thepatient, and other conditions, using an index of the efficacy of eachtherapeutic agent, such as the specificity of effects on infections withP. falciparum, P. vivax, P. malariae, P. ovale, or on complex infectionswith two or more species of these malaria parasites, and furtherchecking the pathogen information including the source of malariainfection and other conditions of the patient with a fever, against themalaria therapeutic agent information stored in the “treatment guidedatabase” including, for example, the effects of drugs, drug names,generic names, areas in which respective drugs are preferably applied,specificity of action, administration periods, dosages, types andfrequencies of side effects, severity of side effects,contraindications, information about infants and pregnant women,information about concomitant use with other drugs, drug prices, etc.

According to the anti-malaria measure support system of the presentinvention, clinical practitioners and hospital doctors can checkinformation about the pathogen of malaria infection in a patient with afever against the malaria therapeutic agent guide database in a PC orvia a cellular phone to have a conventional software to operate, therebyobtaining a display of which malaria therapeutic agent should be givenpriority in selection, or which malaria therapeutic agents should beused in combination.

The above-mentioned malaria therapeutic agent information can be storedin the “treatment guide database”, referring to the Japan HealthSciences Foundation, policy innovative drug development general researchproject, research group on chemotherapy of tropical diseases; theGuidance for Parasitic Disease Chemotherapy, Revised Version 6.0 (2007);the guideline of Expert Meeting on Malaria Chemoprophylaxis published onMarch, 2005; and the malaria treatment guideline of the CDC (Centers forDisease Control and Prevention) in the U.S.

For example, since increasing numbers of P. falciparum strains havebecome chloroquine-resistant, concomitant administration of mefloquineand artesunate is considered the malaria treatment to be given priorityin selection in Thailand and developed countries. On the other hand, inareas where drug prices are reflected, second-generation drugs offansidar take priority in selection in some cases. Against P. vivax andP. ovale, chloroquine takes priority in selection in the erythrocyticstage that causes clinical symptoms such as a fever, and then a two-weekcourse of primaquine administration is selected for complete cure andrecurrence prevention. Further, priority is given to the selection ofchloroquine for use against P. malariae.

Such information is also stored in the “malaria infection-preventionpublic health measure guide database”.

The information about the results of the malaria treatment of a patientwith a fever with the drug selected as a malaria therapeutic agent to begiven priority in selection, using the anti-malaria measure supportsystem of the present invention, is fed back to the “malaria parasitetreatment guide database” and the “malaria infection-prevention publichealth measure guide database”, and stored as updated information.

Thus, an anti-malaria measure support system can be provided in whichinformation about the pathogen of malaria infection in a patient with afever can be obtained easily, rapidly, and with high sensitivity andhigh specificity, by subjecting a specimen extracted from a patient witha fever in a malaria endemic area, or a patient with a fever in anon-endemic area, to the method for identifying infection with a singleor a plurality (complex) of four species of malaria parasites accordingto the present invention; and in which a clinical practitioner or ahospital doctor in a malaria endemic area or non-endemic area can checksuch information against the malaria parasite treatment guide databaseto select a preferable therapeutic agent and administration method thatshould be given priority in selection, for a patient who has developed afever due to malaria infection, and can apply such a drug and method tothe treatment of the malaria-infected patient with a fever.

From the overall viewpoint of malaria infection-prevention public healthmeasures in a malaria endemic area and the treatment of amalaria-infected patient with a fever in a malaria endemic area ornon-endemic area, the present invention can also provide an anti-malariameasure support system for malaria eradication in the endemic area, bycombining: the obtainment of malaria-infected patient information in amalaria endemic area using the method for detecting malaria parasitesaccording to the present invention; the obtainment of information aboutthe pathogen of malaria infection in a patient with a fever in a malariaendemic area or non-endemic area using the anti-malaria measure supportsystem for local governments, autonomous bodies, or residents in amalaria endemic area, in which a malaria infection-prevention publichealth measure database is used, and the method or kit for identifyinginfection with a single or plurality of four species of malariaparasites according to the present invention; and an anti-malariameasure support system in which a clinical practitioner or a hospitaldoctor in a malaria endemic area or non-endemic area using the malariaparasite treatment guide database can select a preferable therapeuticagent and administration method that should given priority in selectionfor a malaria-infected patient with a fever and apply the drug andmethod to the treatment of the malaria-infected patient with a fever.

The above pieces of information can be inputted and stored using a PC,and can be displayed through a PC or a cellular phone, or through apaper brochure, in the area.

The present invention can further provide a malaria infection-preventionmeasure system for travelers from a malaria non-endemic area to amalaria endemic area. The present invention can provide a malariainfection-prevention measure system for persons who plan to travel to amalaria endemic area, the system comprising means for obtaining, fromthe anti-malaria measure support system described above, the state ofthe implementation of public health measures in the malaria endemicarea, information about the pathogens of malaria infection in theendemic area, and the state of the treatment of infected patients; meansfor selecting a malaria prophylactic/therapeutic agent from a malariaparasite treatment guide database; and means for administering theselected agent before travel.

The above pieces of information are easily available from Internetwebsites using a PC or a cellular phone, or as information provided inbrochures from the malaria endemic area or national governmentalorganizations. Using the malaria infection-prevention measure system forpersons who plan to travel to a malaria endemic area, it is possible forpersons who plan to travel to a malaria endemic area to know beforehandthe malaria infections that are endemic in the area and the state of theappearance of drug-resistant strains. Thus, to prevent malariainfection, the persons can take, before travel, a preferable malariatherapeutic agent to be given priority in selection for use against themalaria infections that are endemic in the area, so that even if thepersons should be infected with malaria, symptoms due to malariainfection, such as fever, can be reduced at an early stage and seriousconditions can be prevented, making it possible to exterminate malariaparasites in the blood of such persons at an early stage.

The present invention can further provide a malariainfection-prevention/treatment measure system for returnees from amalaria endemic area, the system comprising: means for obtaining, fromthe anti-malaria measure support system, the state of the implementationof public health measures in the malaria endemic area, information aboutthe pathogens of malaria infection in the endemic area, and the state ofthe treatment of infected patients; means for selecting a malariaprophylactic/therapeutic agent from a malaria parasite treatment guidedatabase; and means for administering the selected agent after returningfrom the malaria endemic area. According to the present invention, sincethe latent period before the appearance of clinical symptoms, such as afever, varies from 1 week to 40 days depending on the species of theinfecting malaria parasites, when a returnee has been infected withmalaria parasites immediately before returning from a malaria endemicarea and in the case where the returnee is aware of having been bittenby a mosquito immediately before returning, a preferable malariatherapeutic agent against malaria parasites that may cause infection canbe selected and administered referring to information from theanti-malaria measure support system, so that symptoms due to malariainfection, such as a fever, can be reduced at an early stage and seriousconditions can be prevented even if the returnee has been infected withmalaria, thereby making it possible to exterminate malaria parasites inthe blood of the returnee from a malaria endemic area at an early stage.

The present invention can also provide a malariainfection-prevention/treatment measure system for returnees from amalaria endemic area, in which, in the above-mentioned malariainfection-prevention/treatment measure system for returnees from amalaria endemic area, when a returnee from a malaria endemic area has afever, the identification of P. falciparum, P. vivax, P. malariae, or P.ovale is performed to select and administer a malaria therapeutic agentthat should be given priority in selection.

Further, the use of the malaria parasite detection/identification methodof the present invention in the malaria infection treatment measuresystem of the present invention makes it possible to monitor thetherapeutic effects of malaria therapeutic agent administration tomalaria-infected patients.

In the above, when the returnee develops a fever before administrationof a malaria therapeutic agent, a specimen from the returnee with afever is subjected to the method for identifying four species of malariaparasites according to the present invention, to identify P. falciparum,P. vivax, P. malariae, P. ovale, or a plurality (complex) thereof, andselect and administer a malaria therapeutic agent that should be givenpriority in selection for use against the malaria parasites. The malariainfection treatment measure system of the present invention can thusprovide a suitable malaria infection treatment method, and a method formonitoring the therapeutic effects thereof.

As shown in FIG. 3, information about the results of public healthmeasures, information about the results of malaria treatment forpatients with a fever, and information about the results ofprophylactic/therapeutic agent administration are fed back intorespective databases.

EXAMPLES

The following Examples illustrate the present invention in furtherdetail, but are not intended to limit the scope of the presentinvention.

Example 1 Materials and Methods

Sixty-eight samples that were positive for malaria parasites bymicroscopy were collected from patients who had visited malaria clinicsin Mae Sod and Mae Kasa, northwestern Thailand. In addition, 53 samplesthat were negative by microscopy were collected from residents in amalaria endemic area of Kanchanaburi, western Thailand.

The blood samples were tested by microscopy and LAMP. Each test wascarried out by independent researchers (microscopy in the Armed ForcesResearch Institute of Medical Sciences, Thailand, and LAMP in EhimeUniversity, Japan), blinded to the origin of the specimens and thelaboratory results, and finally the test results were compared andanalyzed.

Microscopy:

Thick peripheral blood smears were examined under 1,000× magnificationby microscopists with extensive experience in the identification ofmalaria parasites. The parasite density was counted per 500 leukocytesand was then calculated as the number of parasites per microliter byassuming a leukocyte count of 7,000/μl. The initial thick film wasconsidered negative if no parasites were seen after 500 leukocytes werecounted.

DNA Extraction:

The DNA template for LAMP was prepared as described in Plowe, C., et al.(Am. J. Trop. Med. Hyg. (1995) Vol. 52: 565-568). Twenty-five to fiftymicroliters of the human blood was blotted as single spot and dried onfilter paper. A single blood-spot from each filter paper was excised,then incubated for 4 hours at room temperature and/or overnight at 4° C.in 1 ml of 0.5% saponin in phosphate-buffered saline (PBS). The filterpaper was washed for 30 minutes in PBS at 4° C. and transferred into newtubes containing 200 μl of 5% CHELEX-100 cation exchange resin(Bio-Rad,Hercules, Calif.), and vortexed for 30 seconds. The mixture wasincubated at 56° C. for 15 minutes, vortexed for 30 seconds, and heatedat 100° C. for 15 minutes to elute the DNA, vortexed, and centrifuged(10,000×g for 5 minutes). The supernatant was either used immediatelyafter the reaction, or stored in aliquots at −20° C.

LAMP Conditions:

LAMP primer sets for P. falciparum described in Poon et al. (Non-PatentDocument 1) were used. The remaining Plasmodium genus- andspecies-specific LAMP primer sets were attempted to be designed usingthe LAMP Primer Designing Software PrimerExplorer V3 (manufactured byFujitsu Ltd.). However, the nucleotide sequences of the genes of malariaparasites are generally greatly different from those of organisms ofother species and have a high AT content; therefore, it was impossibleto find optimal primer sets using the above primer designing software.Accordingly, many trials and errors were necessary, encounteringdifficulties in designing the primers. Finally, however, primer setshaving sensitivity and specificity sufficient for practical use wereable to be found among the synthesized primer sets with numerouscombinations. Thus, a genus-specific primer set that is capable ofdetecting the four species of the genus Plasmodium that infect humans ata time, and primer sets each specific to each of the four species ofmalaria parasites (P. falciparum, P. vivax, P. malariae, and P. ovale)were successfully designed based on the genus- and species-specificnucleotide sequences of the 18S rRNA genes.

After the nucleotide sequences of the oligonucleotides were designed asabove, primers were synthesized by a known method, for example, usingAutomated DNA Synthesizer, manufactured by Perkin-Elmer.

The location and nucleotide sequence of each primer are shown in FIG. 1.

More specifically, primer sets for the genus Plasmodium and primer setsfor the respective four Plasmodium species were used:

primer sets for the genus Plasmodium [(F3 (SEQ ID NO: 1), B3c (SEQ IDNO: 2), FIP(F1c-F2)(SEQ ID NO: 3), BIP(B1-B2c)(SEQ ID NO: 4), LPF(SEQ IDNO: 5), LPB(SEQ ID NO: 6)];

primer sets for P. falciparum [F3 (SEQ ID NO: 7), B3c (SEQ ID NO: 8),FIP(F1c-F2)(SEQ ID NO: 9), BIP(B1-B2c)(SEQ ID NO: 10), LPF(SEQ ID NO:11), LPB(SEQ ID NO: 12)];

primer sets for P. vivax [F3 (SEQ ID NO: 13), B3c (SEQ ID NO: 14),FIP(F1c-F2)(SEQ ID NO: 15), BIP(B1-B2c)(SEQ ID NO: 16), LPF(SEQ ID NO:17), and LPB(SEQ ID NO: 18)];

primer sets for P. malariae [F3 (SEQ ID NO: 19), B3c (SEQ ID NO: 20),FIP(F1c-F2)(SEQ ID NO: 21), BIP(B1-B2c)(SEQ ID NO: 22), LPF (SEQ ID NO:23), LPB (SEQ ID NO: 24)];

primer sets for P. ovale [F3 (SEQ ID NO: 25), B3c (SEQ ID NO: 26),FIP(F1c-F2)(SEQ ID NO: 27), BIP(B1-B2c)(SEQ ID NO: 28), LPF (SEQ ID NO:29), LPB (SEQ ID NO: 30)].

The LAMP reaction was performed with a Loopamp DNA amplification kit(Eiken Chemical Co., Ltd., Tokyo, Japan).

Reaction mixtures (25 μl) contained 1.6 to 2.4 μM of each FIP and BIP,0.2 μM of each F3 and B3c, 0.8 μM of each LPF and LPB, 2× reaction mix(12.5 μl), Bst DNA polymerase (1 μl), and 1 to 2 μl of DNA sample(corresponding to approximately 0.125 to 0.5 μl of blood).

The LAMP reaction was performed at 60° C. for 100 minutes, then theenzyme was inactivated at 80° C. for 2 minutes.

Analysis of LAMP Products:

The LAMP reaction causes turbidity in the reaction tube, proportional tothe amount of amplified DNA. Therefore, the turbidity was observed withthe naked eye. To confirm the sensitivity of LAMP, turbidity was alsomonitored by a Loopamp real-time turbidimeter (RT-160C, Eiken ChemicalCo., Tokyo, Japan).

For further confirmation, 5 μl of LAMP product was electrophoresed at100 V in a 3% agarose gel, followed by staining with ethidium bromide,using MassRuler™ DNA ladder marker (Fermentas Inc., Hanover, Md.). Thespecificity of LAMP was evaluated by restriction enzyme digestion of theamplified product.

Based on the restriction enzyme maps of the target sequences of eachLAMP product, restriction enzyme, DdeI was selected for restrictionenzyme analysis of the Plasmodium genus-specific LAMP products, HpyCH4Vfor P. falciparum, P. vivax, and P. malariae, and restriction enzyme,AluI for P. ovale. Following overnight digestion at 37° C., the digestedproducts were analyzed by agarose gel electrophoresis.

Diagnostic Threshold of LAMP Results:

The formation of LAMP reaction products was monitored using a Loopampreal-time turbidimeter. Most of the positive samples tested multipletimes showed positivity within 1 hour. Therefore, a sample havingturbidity greater than or equal to the threshold value by turbidimeterwithin 1 hour was considered positive.

Positive Control Plasmid DNA and Sequencing:

For sensitivity assessment, plasmids containing the target region of the18S rRNA gene for LAMP reaction were constructed for each species.Target DNA sequence was amplified with two LAMP primers (F3 and B3c) byPCR, then cloned into the pCR® 2.1-TOPO TA Cloning vector (Invitrogen,Carlsbad, Calif.).

The nucleotide sequences were determined using an automated DNAsequencer (ABI PRISM® 310 Genetic Analyzer, Applied Biosystems).

Analytical Sensitivity and Specificity of LAMP:

To establish the minimum copy number (lower detection limit) of targetgene sequence detectable by LAMP, positive control plasmid DNAs wereused as templates. The standard curve for LAMP was constructed using10-fold serial dilutions of plasmid DNA (10⁶ to 1 copy) into sterilewater. For each standard, the copy number was plotted against thethreshold time. The resulting plots were analyzed by linear regression,and the statistical significance of γ² values was analyzed by ANOVA(Free Statistics and Forecasting Software v1.1.21). Probabilities lessthan 0.05 were considered statistically significant. The specificity ofthe genus- and species-specific LAMP was evaluated on each controlplasmid DNA and P. falciparum genomic DNA (gDNA) purified from NF54strain, P. vivax gDNA from Sal-I strain, P. malariae gDNA from Ugandastrain, and P. ovale CDC type gDNA from CDC strain.

Results of Analytical Sensitivity and Specificity of Plasmodium Genus-and Species-Specific LAMP:

The sensitivity of LAMP for the genus Plasmodium and four species ofmalaria parasite, P. falciparum, P. vivax, P. malariae, and P. ovale,was 10 copies for P. malariae and P. ovale, and 100 copies for the genusPlasmodium, P. falciparum, and P. vivax.

The specificity of each LAMP reaction was further confirmed byrestriction enzyme digestion of LAMP products. The sizes of theresultant digestion products were in good agreement with the predictedsizes.

Clinical Sensitivity and Specificity:

The clinical sensitivity and specificity of the Plasmodium LAMP werecalculated on 121 whole-blood samples with microscopy as the referencestandard method. Sensitivity was calculated as (number of truepositives)/(number of true positives+number of false negatives), andspecificity was calculated as (number of true negatives)/(number of truenegatives+number of false positives).

Clinical Sensitivity and Specificity: Comparison of Microscopy and LAMP:

The results of microscopy and LAMP are given in Table 2.

Among 68 patients who were positive for malaria parasite by microscopy,12 patients were diagnosed with P. falciparum infection, 34 with P.vivax infection, 12 with P. malariae infection, 5 with P. ovaleinfection, and 5 with mixed P. falciparum and P. vivax infection. Theremaining 53 samples were negative by microscopy.

LAMP using the genus-specific primer set detected malaria parasites in67 samples out of 68 samples positive by microscopy (98.5% sensitivity).Among the 53 samples that were negative by microscopy, genus-specificLAMP detected malaria parasites in 3 samples (94.3% specificity).

The 3 samples positive by LAMP but negative by microscopy were re-testedby LAMP.

All three samples were again positive by genus-specific LAMP; one wasdiagnosed with P. falciparum and the other two with P. vivax byspecies-specific LAMP.

It is thus presumed that LAMP is more sensitive than microscopy and canreduce the false negative diagnosis, which may clinically causeoversights, to the least possible degree.

All 12 samples that were positive for P. falciparum by microscopy werealso positive for P. falciparum by species-specific LAMP. Among 34samples positive for P. vivax by microscopy, 2 samples were diagnosedwith P. ovale infection, and 1 with mixed P. falciparum and P. vivaxinfection, by LAMP. Among 12 samples positive for P. malariae, 1 samplewas diagnosed with P. ovale, and 1 sample with mixed P. malariae and P.vivax.

The above results indicate that the new LAMP malaria diagnosis methoddeveloped by the present inventors has higher sensitivity and higherspecificity than microscopy, and is an advantageous method for detectingthe four species of human malaria parasites.

The average detection time by LAMP was as follows.

Genus-specific LAMP: 25.7±4.9 minutes (mean±SD; 19.4 to 52.9 minutes);

P. falciparum-specific LAMP: 31.7±4.8 minutes (25.8 to 44.9 minutes);

P. malariae-specific LAMP: 30.6±5.2 minutes (25.4 to 46.9 minutes);

P. vivax-specific LAMP: 34.8±4.8 minutes (30.5 to 46.6 minutes); and P.ovale-specific LAMP: 36.1±6.8 minutes (29.9 to 49.8 minutes).

These results show that LAMP can make diagnosis in a period of time thatis remarkably shorter than the amplification time of nested PCR.

Example 2

The primer sets for detecting the genus Plasmodium and Plasmodiumspecies according to the present invention, and the method for detectingor identifying the genus Plasmodium, P. falciparum, P. vivax, P.malariae, and P. ovale separately or simultaneously using the primersets, were subjected to comparative tests with PCR and microscopy in amalaria clinic in Mae Sod, northwestern Thailand, where malaria isendemic.

A comparison was also made between the above method carried out usingequipment specially designed for LAMP, and the above method carried outby a procedure that can be rapidly and easily performed in a malariaendemic area.

Eighteen samples that were positive for malaria parasites by microscopy(the percent parasitemia: 0.04% to 0.31%) were used; and one sample thatappeared to be a technical error was excluded from the Examplebeforehand.

Boiling method was used to extract DNA from patient samples for therapid and easy detection and identification of malaria parasites in amalaria endemic area, according to the present invention (the genusPlasmodium, P. falciparum, P. vivax, P. malariae, and P. ovale can bedetected simultaneously). Specifically, 50 μl of distilled water (D.W.)was added to 50 μl of a blood sample, and the resulting mixture wasboiled at 99° C. for 5 minutes, followed by centrifugation (5415D,produced by Eppendorf; 16,000×g) to obtain a supernatant.

Subsequently, 2 μl of the above-obtained supernatant containing DNA wasadded to 23 μl of a reaction mixture containing 1 μl of Bst DNApolymerase (Epicentre Biotechnologies), 12.5 μl of 2× reaction mix, 1.6to 2.4 μM of FIP and BIP in the primer sets obtained in Example 1, 0.2μM of F3 and B3c in the primer sets obtained in Example 1, and 0.8 μM ofLPF and LPB in the primer sets obtained in Example 1, and reacted at 60°C. for about 60 minutes in a constant-temperature water bath(Thermominder SM-05R, produced by TAITEC) capable of containing 96×2(total 192) tubes.

The presence or absence of turbidity in the reaction product wasvisually observed directly to detect the presence or absence ofinfection with the genus Plasmodium and identify which of the P.falciparum, P. vivax, P. malariae, or P. ovale causes the infection.

Separately, the reaction mixture containing DNA extracted from each ofthe above samples was reacted at 60° C. for about 60 minutes using aLoopamp real-time turbidimeter (LA-320C, produced by Eiken Chemical Co.,Ltd.) to monitor the formation of the reaction product in real time.

Further, for comparison with PCR with respect to the diagnosis ofmalaria species, nested PCR was performed on the same samples. Sincestrict conditions are required for a PCR reaction, the PCR reaction maybe inhibited when DNA extracted from a clinical sample by boiling methodis used as it is. Thus, dried blood on filter paper obtained in themalaria clinic was brought to a well-equipped laboratory in Bangkok, andDNA was extracted using a DNA extraction kit (QIAamp DNA Mini Kit,produced by QIAGEN) and subjected to PCR. In the nested PCR, two roundsof PCR were each performed for 2 hours, for a total of 4 hours.Thereafter, the obtained sample was subjected to agarose gelelectrophoresis and stained with fluorescence, and detection wasperformed for a total of 5 hours.

As a result, of the 18 samples that were positive for malaria parasitesby microscopy, in the detection of the presence or absence of infectionwith the genus Plasmodium using the primers for the genus Plasmodium, 18were positive when the samples were tested using the Loopamp real-timeturbidimeter specially designed for LAMP, and 18 were also positive whenthe samples were tested by reacting in a constant-temperature water bathfollowed by visual observation. The results of the two tests agreed100%.

Table 1 compares the test results for microscopy, nested PCR, and LAMPfor the diagnosis of malaria species using Plasmodium species-specificprimers; and the results using an amplifier specially designed for LAMP,with the results of the visual observation after the reaction in aconstant-temperature water bath.

TABLE 1 LAMP Constant- temperature Sample Specially water bath +(Percent designed visual parasitemia) PCR amplifier observation Pf(0.12-0.28%) +  3 (N.D.2) 5 5 Pv (0.04-0.31%) + 12 11 11 Pv + Po Pv+  11 1 (00.8, 0.04%) Po−  1 1 1

As is apparent in Table 1, with respect to P. falciparum, only 3 sampleswere subjected to PCR. In the table, Pf indicates P. falciparum, Pvindicates P. vivax, and Po indicates P. ovale.

As a result, in one sample diagnosed by microscopy as being infectedwith both P. vivax and P. ovale, both PCR and LAMP (both the test usingan amplifier specially designed for LAMP, and the test using visualobservation after the reaction in a constant-temperature water bath)detected P. vivax, but not P. ovale, indicating that the microscopydiagnosis was an error.

That is, in the comparative test, 17 of the 18 LAMP reaction resultsmatched the microscopy results (94% agreement). This means that the LAMPreaction results agreed 100% with the microscopy results, if theabove-mentioned one reaction result in disagreement with the microscopyresult is excluded. These results reveal that the primer sets fordetecting the genus Plasmodium and Plasmodium species according to thepresent invention, and the method for detecting or identifying the genusPlasmodium, P. falciparum, P. vivax, P. malariae, and P. ovaleseparately or simultaneously using these primers, agreed 100% with themicroscopy diagnoses in the detection and identification of Plasmodiumparasite infection; and the test results using an amplifier speciallydesigned for LAMP agreed 100% with the visual observation results afterthe reaction in the constant-temperature water bath. In the diagnoses ofmalaria species, the primer sets and method agreed 100%, and achievedsensitivity equivalent to PCR.

It was demonstrated that, for the primer sets for detecting the genusPlasmodium and Plasmodium species according to the present invention,and the method for detecting or identifying the genus Plasmodium, P.falciparum, P. vivax, P. malariae, and P. ovale separately orsimultaneously using the primer sets, especially in a malaria endemicarea, DNA can be extracted by boiling method, which is rapid, simple,and inexpensive, and a DNA amplification reaction can be carried outusing a constant-temperature water bath so that a large number ofsamples can be treated inexpensively.

The primer sets for detecting the genus Plasmodium and Plasmodiumspecies according to the present invention and the method for detectingor identifying the genus Plasmodium, P. falciparum, P. vivax, P.malariae, and P. ovale separately or simultaneously using the primersets, can be applied more rapidly and easily than microscopy, and havesensitivity equivalent to PCR. Therefore, the primer sets and method canbe advantageously applied on-site, especially in a malaria endemic area,for the rapid and simple detection and identification diagnosis of thePlasmodium genus and species simultaneously, in a large number ofsamples.

Example 3

According to the present invention, from the test results obtainedon-site (in a clinic) in a malaria endemic area in a short period oftime, mefloquine and artesunate can be prescribed for administration tothe five patients diagnosed and identified as being infected with P.falciparum in Example 2, based on the information about malariatherapeutic agents and the priority thereof in the malaria therapeuticagent information stored in the “treatment guide database”. Likewise,chloroquine and primaquine can be prescribed for administration to the13 patients diagnosed and identified as being infected with P. vivax.Thus, an anti-malaria measure support system can be operated which iscapable, in a malaria endemic area, of rapidly and reliably detectingthe presence or absence of infection with Plasmodium parasites andproviding malaria treatment.

Example 4

As described above, simple and easy detection of the genus Plasmodium(in particular, the presence or absence of mixed infection) is importantin malaria endemic areas.

The present inventors conducted further research on primers that arecapable of detecting Plasmodium species.

As a result, the inventors found that two types of primer sets (Pv-7 andPv-9; Pv-7 is represented by SEQ ID NOs: 37 to 42 and Pv-9 isrepresented by SEQ ID NOs: 31 to 36) that are useful for P. vivaxdiagnosis enable more rapid diagnosis of P. vivax.

When using the two types of primer sets, it was demonstrated that theseprimer sets react none of the DNAs of P. falciparum, P. malariae, and P.ovale.

The use of the two types of primer sets (Pv-7 and Pv-9) useful for P.vivax diagnosis achieved more rapid diagnosis (Pv-7: 27 minutes; Pv-9:24 minutes) than the previously found primer set for P. vivax diagnosis(31 minutes). In particular, the use of the primer set Pv-9 achieved themost rapid diagnosis.

The invention further provides the following inventions:

1A. A primer set for detecting the genus Plasmodium, comprising anoligonucleotide set containing nucleic acid sequences represented by SEQID NOs: 1 to 6, the primer set being capable of amplifying a particularregion of a Plasmodium 18S rRNA gene sequence.

2A. A primer set for detecting Plasmodium vivax, comprising anoligonucleotide set containing nucleic acid sequences represented by SEQID NOs: 13 to 18, the primer set being capable of amplifying aparticular region of a Plasmodium vivax 18S rRNA gene sequence.

3A. A primer set for detecting Plasmodium malariae, comprising anoligonucleotide set containing nucleic acid sequences represented by SEQID NOs: 19 to 24, the primer set being capable of amplifying aparticular region of a Plasmodium malariae 18S rRNA gene sequence.

4A. A primer set for detecting Plasmodium ovale, comprising anoligonucleotide set containing nucleic acid sequences represented by SEQID NOs: 25 to 30, the primer set being capable of detecting a particularregion of a Plasmodium ovale 18S rRNA gene sequence.

5A. A primer set for detecting the genus Plasmodium, P. falciparum, P.vivax, P. malariae or P. ovale, comprising an oligonucleotide primer setcontaining any of the nucleic acid sequences of claims 1A to 4A andnucleic acid sequences represented by SEQ ID NOs: 7 to 12, the primerset being capable of amplifying particular regions of a 18S rRNA gene ofthe genus Plasmodium and various species of Plasmodium including aparticular region of a Plasmodium falciparum 18S rRNA gene sequence.

6A. A method for detecting the genus Plasmodium present in a specimen,wherein the method targets a particular region of the genus Plasmodium18S rRNA gene sequence, and comprises selectively amplifying theparticular region of the genus Plasmodium 18S rRNA gene sequence by LAMPusing a primer set of claim 1A, and confirming the presence or absenceof an amplified product.

7A. A method for detecting Plasmodium vivax present in a specimen,wherein the method targets a particular region of a Plasmodium vivax 18SrRNA gene sequence, and comprises selectively amplifying the particularregion of the Plasmodium vivax 18S rRNA gene sequence by LAMP using aprimer set of claim 2A, and confirming the presence or absence of anamplified product.

8A. A method for detecting Plasmodium malariae present in a specimen,wherein the method targets a particular region of a Plasmodium malariae18S rRNA gene sequence, and comprises amplifying the particular regionof the Plasmodium malariae 18S rRNA gene sequence by LAMP using a primerset of claim 3A, and confirming the presence or absence of an amplifiedproduct.

9A. A method for detecting Plasmodium ovale present in a specimen,wherein the method targets a particular region of a Plasmodium ovale 18SrRNA gene sequence, and comprises selectively amplifying the particularregion of the Plasmodium ovale 18S rRNA gene sequence by LAMP using aprimer set of claim 4A, and confirming the presence or absence of anamplified product.

10A. A method for detecting the genus Plasmodium, Plasmodium falciparum,Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale present in aspecimen, wherein the method targets a particular region of the genusPlasmodium 18S rRNA gene sequence, the genus Plasmodium falciparum 18SrRNA gene sequence, a Plasmodium vivax 18S rRNA gene sequence, aPlasmodium malariae 18S rRNA gene sequence, or a Plasmodium ovale 18SrRNA gene sequence;

and wherein the method comprises selectively amplifying the particularregion of the genus Plasmodium 18S rRNA gene sequence, P. falciparum 18SrRNA gene sequence, P. vivax 18S rRNA gene sequence, P. malariae 18SrRNA gene sequence, or P. ovale 18S rRNA gene sequence respectively; byLAMP using a primer set of claim 5A; and confirming the presence orabsence of an amplified product.

11A. A detection method according to claim 10A, wherein the genusPlasmodium, P. falciparum, P. vivax, P. malariae or P. ovale aredetected simultaneously or separately.

12A. A method for identifying the genus Plasmodium, comprising isolatinga DNA sample from a specimen, performing LAMP amplification of aparticular region of the genus Plasmodium 18S rRNA gene sequence fromthe DNA sample using a primer set of claim 1A, and confirming thepresence or absence of an amplified product.

13A. A method for identifying Plasmodium vivax, comprising isolating aDNA sample from a specimen, performing LAMP amplification of aparticular region of a Plasmodium vivax 18S rRNA gene sequence from theDNA sample using a primer set of claim 2A, and confirming the presenceor absence of an amplified product.

14A. A method for identifying Plasmodium malariae, comprising isolatinga DNA sample from a specimen, performing LAMP amplification of aparticular region of a P. malariae 18S rRNA gene sequence from the DNAsample using a primer set of claim 3A, and confirming the presence orabsence of an amplified product.

15A. A method for identifying Plasmodium ovale, comprising isolating aDNA sample from a specimen, performing LAMP amplification of aparticular region of a P. ovale 18S rRNA gene sequence from the DNAsample using a primer set of claim 4A, and confirming the presence orabsence of an amplified product.

16A. A method for identifying the genus Plasmodium, P. falciparum, P.vivax, P. malariae, or P. ovale, comprising isolating a DNA sample froma specimen, performing LAMP amplification of a particular region of thegenus Plasmodium 18S rRNA gene sequences, a P. falciparum 18S rRNA genesequence, a P. vivax 18S rRNA gene sequence, a P. malariae 18S rRNA genesequence, or a P. ovale 18S rRNA gene sequence, from the DNA sampleusing a primer set of claim 5A; and confirming the presence or absenceof an amplified product.

17A. A method of identification according to claim 16A, wherein theidentification of any of the genus Plasmodium, P. falciparum, P. vivax,P. malariae, or P. ovale is performed simultaneously or separately.

18A. A detection kit for the genus Plasmodium, P. falciparum, P. vivax;P. malariae, or P. ovale; comprising at least a primer set of any ofclaim 1A to 5A, a strand displacement DNA polymerase, dNTPs and areaction buffer.

19A. A detection kit according to claim 18A, wherein the detection kitdetects the genus Plasmodium, P. falciparum, P. vivax, P. malariae, orP. ovale, simultaneously or separately.

20A. An anti-malaria measure support system comprising:

means for inputting and storing malaria-infected patient informationincluding the number positive for the genus Plasmodium parasites thatcause malaria in a malaria endemic area, and the carrier rate in thearea;

a malaria infection-prevention public health measure guide database thatspecifies public health measures for the malaria endemic area based onthe malaria-infected patient information, into which database publichealth measure selection information for a malaria parasite-infectedarea to be inputted together with the measure priority indicating whichof the public health measures should be given priority in selection hasbeen inputted;

a public health measure extraction section that extracts public healthmeasures for the malaria-infected endemic area and the priority thereoffrom the malaria infection-prevention public health measure guidedatabase, according to malaria-infected patient information aboutmalaria parasites in a subject; and

a public health measure display section that displays the public healthmeasures extracted in the public health measure extraction section,together with the priority thereof.

21A. An anti-malaria measure support system according to claim 20A,wherein the malaria-infected patient information about malaria parasitesin the malaria endemic area is obtained by identifying the presence orabsence of infection with the genus Plasmodium using a primer setaccording to claim 1A and/or a detection method according to claim 6A,or the genus Plasmodium detection kit according to claim 19A.

22A. An anti-malaria measure support system comprising:

means for inputting and storing malaria therapeutic agent informationfor specifying a malaria therapeutic agent that acts on infection withone or a plurality of four species of malaria parasites;

patient information input means for inputting and storing patientinformation including information about the pathogen of malariainfection in a patient with a fever in a malaria endemic area;

patient information input means for inputting and storing patientinformation including information about the pathogen of malariainfection in a patient with a fever in a malaria non-endemic area;

a treatment guide database into which, according to indices of efficacyagainst malaria parasites detected in a subject, malaria therapeuticagent selection information to be inputted together with a priority thatindicates which malaria therapeutic agent should be given priority inselection for use against the malaria parasites;

a malaria therapeutic agent extraction section that extracts, accordingto the information about the pathogen of malaria infection in thesubject, malaria therapeutic agents to be administered and the prioritythereof, from the treatment guide database; and

a malaria therapeutic agent display section that displays the malariatherapeutic agents extracted in the above malaria therapeutic agentextraction section, together with the priority thereof.

23A. An anti-malaria measure support system according to claim 22A,wherein the information about the pathogen of malaria infection in apatient with a fever is obtained by identifying infection with one or aplurality of four species of malaria parasites using a primer setaccording to any one of claims 2A to 5A and/or a method for identifyingthe genus Plasmodium, Plasmodium falciparum, Plasmodium vivax,Plasmodium malariae, or Plasmodium ovale according to any one of claims13A to 17A, or a detection kit according to claim 18A or 19A.

24A. An anti-malaria measure support system in which a public healthmeasure relating to an anti-malaria measure and a malaria parasitetreatment measure relating to an anti-malaria measure are carried out incombination;

the public health measure comprising:

means for inputting and storing malaria-infected patient informationincluding the number positive for the genus Plasmodium that causesmalaria in a malaria endemic area, and the carrier rate in the area;

a malaria infection-prevention public health measure guide database thatspecifies public health measures for the malaria endemic area based onthe malaria-infected patient information, into which database publichealth measure selection information for a malaria parasite-infectedarea to be inputted together with a priority of measures that indicateswhich of the public health measures should be given priority inselection has been inputted;

a public health measure extraction section that extracts public healthmeasures for the malaria-infected endemic area and the priority thereof,from the malaria infection-prevention public health measure guidedatabase according to malaria-infected patient information about malariaparasites in the subject; and

a public health measure display section that displays the public healthmeasures extracted in the public health measure extraction section,together with the priority thereof; and

the malaria parasite treatment measure comprising:

means for inputting and storing malaria therapeutic agent informationfor specifying a malaria therapeutic agent that acts on infection withone or a plurality of four species of malaria parasites;

patient information input means for inputting and storing patientinformation including information about the pathogen of malariainfection in a patient with a fever in the malaria endemic area;

patient information input means for inputting and storing patientinformation including information about the pathogen of malariainfection in a patient with a fever in a malaria non-endemic area;

a treatment guide database into which, according to indices of efficacyagainst malaria parasites detected in a specimen, malaria therapeuticagent selection information to be inputted together with a priority thatindicates which malaria therapeutic agent should be given priority inselection for use against the malaria parasites;

a malaria therapeutic agent extraction section that extracts, accordingto the information about the pathogen of malaria infection in thesubject, malaria therapeutic agents to be administered and the prioritythereof, from the treatment guide database; and

a malaria therapeutic agent display section that displays the malariatherapeutic agents extracted in the above malaria therapeutic agentextraction section, together with the priority thereof.

25A. An anti-malaria measure support system according to claim 24A,wherein the malaria-infected patient information about malaria parasitesin the malaria endemic area is obtained by identifying the presence orabsence of infection with the genus Plasmodium using a primer setaccording to claim 1A and/or a detection method according to claim 6A,or the genus Plasmodium detection kit according to claim 19A; and/orinformation about the pathogen of malaria infection in a patient with afever is obtained by identifying infection with one or a plurality offour species of malaria parasites using a primer set according to anyone of claims 2A to 5A and/or a method for identifying the genusPlasmodium, Plasmodium falciparum, Plasmodium vivax, Plasmodiummalariae, or Plasmodium ovale according to any one of claims 13A to 17A,or a detection kit according to claim 18A or 19A.

26A. A malaria infection-prevention measure system for persons who planto travel to a malaria endemic area, the system comprising:

means for obtaining the state of the implementation of public healthmeasures in the malaria endemic area, information about the pathogens ofmalaria infection in the endemic area, and the state of the treatment ofinfected patients, from the anti-malaria measure support systemaccording to claim 24A;

means for selecting a malaria prophylactic/therapeutic agent from amalaria parasite treatment guide database; and

means for administering the selected agent before travel.

27A. A malaria infection-prevention/treatment measure system forreturnees from a malaria endemic area, the system comprising:

means for obtaining the state of the implementation of public healthmeasures in the malaria endemic area, information about the pathogens ofmalaria infection in the endemic area, and the state of the treatment ofinfected patients;

means for selecting a malaria prophylactic/therapeutic agent from amalaria parasite treatment guide database; and

means for administering the selected agent after returning from themalaria endemic area, according to Item 24.

28A. A malaria infection-prevention/treatment measure system accordingto claim 27A, wherein, when a returnee from the malaria endemic area hasa fever, identification of Plasmodium falciparum, Plasmodium vivax,Plasmodium malariae, or Plasmodium ovale is performed to select andadminister a malaria therapeutic agent that should be given priority inselection.

INDUSTRIAL APPLICABILITY

The method for detecting/identifying the genus Plasmodium and fourspecies of malaria parasites using LAMP developed by the presentinventors have higher sensitivity and higher specificity thanmicroscopy, and can detect/identify the Plasmodium parasites inspecimens more easily and in a shorter period of time than PCR. Further,since the method is inexpensive, it is very useful for malaria clinicaldiagnosis and control activity in malaria endemic areas with poorfacilities. It is also possible to construct a new anti-malaria measuresupport system (FIG. 3) using the method.

SEQUENCE LISTING FREE TEXT

The 18S rRNA sequences of four Plasmodium species, P. falciparum(GenBank Accession No. M19172), P. vivax (GenBank Accession No. U03079or X13926), P. malariae (GenBank Accession No. M54897), and P. ovale(GenBank Accession No. L48987), were aligned for comparison.

1-27. (canceled)
 28. A method of detecting Plasmodium ovale in abiological sample with a malarial infection, the method comprising: (a)extracting DNA from the biological sample; (b) amplifying a region of aPlasmodium 18S rRNA gene sequence by reacting the DNA extracted in step(a) in a reaction mixture containing a strand displacement DNApolymerase and a sequence-specific primer set which is anoligonucleotide set containing nucleic acid sequences represented by SEQID NOs: 25 to 30; and (c) detecting the presence or absence of anamplified product of step (b); wherein the presence of saidamplification product is indicative of the presence of Plasmodium ovalein the biological sample.
 29. The method according to claim 28, whereinthe DNA extraction from the specimen in step (b) is carried out byboiling the specimen containing the DNA, and performing centrifugation.30. The method according to claim 29, wherein the boiling time is up toten minutes.
 31. The method according to claim 28, wherein theamplification in step (b) of the region of Plasmodium 18S rRNA genesequence is performed at about 60° C. for about 1 hour using aconstant-temperature water bath or an amplifier specially designed forloop-mediated isothermal amplification (LAMP).
 32. The method accordingto claim 28, wherein the presence or absence of the amplificationproduct is identified by visual observation of turbidity or a real-timeturbidimeter.
 33. The method according to claim 28, which is performedin a malaria endemic area.